1. Field of the Invention
This invention relates to the field of cell-free protein synthesis, and more particularly to methods and compositions for in vitro protein synthesis.
2. Description of the Background and Related Art
Cell-free translation systems have been used in the study of protein biosynthesis and have become a standard tool in molecular biology for studying messenger ribonucleic acids (RNAs) and other nucleic acids. Both eukaryotic and prokaryotic cell-free systems have been used for in vitro protein synthesis. The rabbit reticulocyte (Pelham and Jackson, Eur. J. Biochem., 67: 247-256 (1976)) and wheat germ lysate (Roberts and Paterson, Proc. Natl. Acad. Sci., 70: 2330-2334 (1973)) methods are commonly used eukaryotic in vitro translation systems. The E. coli S30 extract method devised by Zubay, Ann. Rev. Genet., 7: 267 (1973) and the fractionated method of Gold and Schweiger, Meth. Enzymol., 20: 537 (1971) are widely used prokaryotic in vitro translation systems.
In the case of the rabbit reticulocyte method, cell-free reticulocyte lysates continue to synthesize protein at approximately 60% of the rate of intact cells for up to one hour. Findeis and Whitesides, Appl. Biochem. Biotechnol., 15: 169-189 (1987) found that the addition of extra adenosine 5'-triphosphate(ATP), guanosine 5'-triphosphate(GTP) and Mg.sup.2+ to the lysates produced a modest gain in protein synthesis, but were unable to reduce the loss of translational activity after 2 hours and concluded that the rabbit reticulocyte method is unsuitable for preparation of gram quantities of product.
Pratt (Pratt, J. M., "Coupled Transcription-Translation in Prokaryotic Cell-Free Systems", in Transcription and Translation: A Practical Approach, Hames and Higgins, eds, IRL Press (1987), pp.179-209) optimized the plasmid deoxyribonucleic acid (DNA) concentration (1.6 .mu.g/.mu.l) and the Mg.sup.2+ concentration(10-15 mM) used in the E. coli extract methods of Zubay (1973) and Gold and Schweiger (1971). However, the E. coli extract methods of Pratt (1987), Zubay (1973), and Gold and Schweiger (1971) produce a 0.3 microgram/milliliter-hour(.mu.g/ml-hr) volumetric rate of protein synthesis, approximately 5,000 fold lower than the volumetric rate achieved by in vivo synthesis of recombinant protein in E. coli hosts.
Lesley et al., J. Biol. Chem., 2: 2632-2638 (1991) optimized the Zubay (1973) E. coli extract for use with PCR fragments and other linear DNA templates by preparing the bacterial extract from a nuclease-deficient strain of E. coli.
Baranov et al., Gene, 84: 463-466 (1989) used a continuous E. coli extract in vitro system to obtain protein synthesis for 20 to 50 hours. In the Baranov et al. (1989) system, the reaction mixture was continuously fed with nucleotide 5'-triphosphates (NTPs), phosphoenolpyruvate (PEP) and amino acids and products were continuously removed from the reaction vessel through ultrafiltration membranes. However, the Baranov et aL system produced a volumetric rate of protein synthesis of less than 4 .mu.g/ml-hr whereas the volumetric rate for in vivo synthesis of a recombinant protein in an E. coli expression host is approximately 1500 .mu.g/ml-hr.
Kudlicki et al., Analyt. Biochem., 206: 389-393 (1992) reported a continuous flow E. coli extract in vitro system using a modification of the Zubay (1973) method in which circular (non-linearized) plasmid DNA is used as the template for protein synthesis and a purified ribosome fraction is used in place of bacterial extract to drive in vitro translation. In the Kudlicki et al. method, the E. coli (S30) extract is prepared according to Zubay (1973) and then the ribosome fraction is isolated from the S30 extract by high speed centrifugation.